The invention is directed to a method and apparatus, which may be in kit form, for accurate and rapid mass processing of large numbers of test samples absorbed onto an absorbent support, such as a nitrocellulose paper support, or other suitable support. The apparatus of the invention is particularly well suited for rapid assaying of large numbers of experimental cell cultures grown in standard tissue culture plates having 96 individual culture wells arranged in 8 rows of 12 wells each.
Sensitive assay systems for screening cell culture test samples for the presence of a specific product are known which entail adsorption of a small quantity of a culture medium onto nitrocellulose paper, or other suitable adsorbent support means, and assay of the absorbed material in situ. Immunoassay methods known in the art for assay of material absorbed on, e.g., nitrocellulose supports, include radioimmunoassay (RIA) and enzyme-linked-immunosorbent assay (ELISA). Still other known assays which are performed on test samples absorbed on a paper support include the use of labeled RNA and DNA probes to assay culture samples absorbed on nitrocellulose for the presence of a complimentary segment of DNA.
When a test sample is to be assayed for the presence of a specific antibody, an immunoassay is performed whereby a radio-labelled antigen or an antigen linked with an enzyme is contacted with the test sample which is absorbed onto the support. Binding of the antigen to the absorbed test sample indicates the presence of the desired antibody product. Binding of the antigen can be conveniently determined in situ by a visible color reaction (ELISA) or by counting the radioactivity (RIA) that is bound to the test sample absorbed on the nitrocellulose, depending upon the type of immunoassay system used. Although these assay systems are reliable and easily performed, they are labor-intensive, and become quite burdensome and expensive when mass screening of many test culture samples is involved.
In current hybridoma methodology, a mouse myeloma cell is fused with a spleen cell of a mouse immunized with a specific antigen in order to obtain an individual cell line that is producing a myeloma antibody to that antigen in tissue culture. One must fuse a large number (10.sup.7 -10.sup.8) of myeloma cells with an equally large population of spleen cells, thus producing heterogeneous antibodies. Of those cells which fuse into immortal hybrid cells ("hybridomas"), only a small fraction produce an antibody to the desired antigen. It is therefore necessary to isolate from the heterogeneous population of experimental hybridomas, the one hybridoma cell which produces a monoclonal antibody to the specific antigen.
In order to isolate the one specific hybridoma cell from the heterogeneous experimental population, the experimental cell culture is distributed among a plurality of wells in tissue culture plates. Typically, these plates have 96 wells per plate. The cells are incubated and divide in their individual wells, and a sample is taken from the culture medium in each well and then assayed for the presence of the specific desired antibody. If a sample from a well tests positive for the specific antibody, the cells are taken up and the population divided by redistribution into other plates of, e.g., 96 wells. The cells are incubated in these wells and the assay is repeated again for the detection of samples in wells containing cells producing the desired antibody. In this manner, antibody-positive wells are repeatedly identified and subcloned, until all the cells in a given well are derived from a single cell, i.e., the cell culture is monoclonal.
This repetitive subcloning and assaying of antibody producing wells to obtain a pure monoclonal hybridoma is tedious and, as heretofore practiced, is highly labor-intensive. A small quantity of test culture sample must be taken from each well and absorbed onto an adsorbent assay support such as nitrocellulose paper, and each absorbed test sample spot must then be assayed by an RIA or ELISA procedure.
In an ELISA system, a small sample is withdrawn from each cell culture well and absorbed onto nitrocellulose paper. This first application provides any antibodies that are being produced in the culture medium. Next, a blocking solution of BSA, gelatin or other solution is applied to block nonspecific protein binding by the paper. To do this, the entire sheet of nitrocellulose paper is submerged in the blocking solution. The third step is the application of the specific antigen to the nitrocellulose paper having the cell culture sample absorbed thereon. The antigen is usually conjugated to an enzyme which can be assayed by a color reaction. If there is an antibody present in the cell culture absorbed onto the nitrocellulose paper, the antibody binds the antigen and thereby also the conjugated enzyme. The fourth step is the application of the substrate for the conjugated enzyme. As a final result of the reaction of the enzyme and its substrate, a measurable color develops on the nitrocellulose paper where the test sample was absorbed. A color reaction is visible on the nitrocellulose paper when the specific antibody to the antigen conjugated to the enzyme is present in the absorbed cell culture test sample. The intensity of the color is proportional to the amount of specific antibody in the cell culture.
To perform an RIA procedure, a sample is withdrawn from a test culture well and absorbed onto nitrocellulose paper, to immobilize any antibodies which might be present. The nitrocellulose paper with absorbed sample is then submerged in a blocking solution. A radio-labelled antigen is then applied to the nitrocellulose paper containing the absorbed test culture sample. The radio-labelled antigen is bound to the test sample only if the specific antibody to the antigen is present in the test sample. Radioactivity bound to the nitrocellulose paper through the test sample is counted in a gamma or liquid scintillation counter.